This invention relates to apparatus for extraction of tetrodotoxin. The apparatus can be used, inter alia, for batch production of tetrodotoxin or other toxins from animal tissues, for example using the method described in another application of the inventors (application Ser. No. 09/695,711, filed Oct. 25, 2000. Twenty to one hundred kilograms of raw material can be processed at one time when puffer fish ovaries are used.
A method for extraction of tetrodotoxin from animal tissues is described in a prior patent application of the inventors (U.S. application Ser. No. 09/695,711, filed Oct. 25, 2000. That method comprises five steps as follows:
Step 1: Grind the tissues into small pieces, soak with an amount of water equal to 1.5 times by weight of the tissues and an amount of a weak organic acid, typically a carboxylic acid, preferably acetic acid, equal to 0.05%-1%, preferably 0.1%-0.3%, by weight of the tissue for several hours, then stir and filter quickly to obtain a lixiviated solution. Repeat this step 3-4 times in order to extract as much toxin as possible.
Step 2: Heat the lixiviated solution to 70-95xc2x0 C. to coagulate and remove soluble proteins (xe2x80x9cscleroproteinxe2x80x9d).
Step 3: Adjust the pH of the lixiviated solution obtained in step 1 to 6.0xcx9c7.5 using an aqueous solution of a weak base, then put the solution through a weakly acidic cation ion-exchange resin to enrich tetrodotoxin. Elute the bound tetrodotoxin with a weak acid.
Step 4: Adjust the pH of the obtained tetrodotoxin solution in step 3 to a range between 8 and 9 for a period of 2-4 hours, during which the solution is put through a column filled with active charcoal and diatomaceous silica so as to remove inorganic salts and a fraction of the alkaline amino acids. Tetrodotoxin binds the immobile phase, which is washed with de-ionized water, then the toxin is eluted with acidic ethanol solution.
Step 5: Purify and crystallize the tetrodotoxin by concentrating the solution obtained in step 4 under vacuum, then adjusting to an alkaline pH. Vacuum dry the obtained tetrodotoxin crystals, typically about 24 hours until the weight of the crystals becomes constant.
The method as described above can be run in either a batch mode or a continuous mode, but is typically run in a batch mode. The extraction system described herein is an efficient system for continuous production. A production cycle can be completed within a period of 6-7 days providing that the extraction is conducted 24 hours a day and 20-100 kg puffer fish ovaries are loaded at one time. For a load of 20 kg material, 0.8-1.2 grams tetrodotoxin can be obtained; for a load of 100 kg, 4-6 grams can be obtained.
The extraction system of this invention is named IWT-112 (See FIG. 1). An important component of the IWT-112 system is a lixiviating and filtering apparatus (xe2x80x9clixiviatorxe2x80x9d, device IWT-113). Additional components are a heater, an ion-exchange column, a diatomaceous silica-active carbon column, a vacuum concentrator etc. The components of the system are arranged so that liquids flow from the outlet of one device into the inlet of the next device. For continuous mode operation, it is possible to arrange the downstream components so that a batch run from the lixiviator is directed to one of several sets of series of the downstream components. Flow through the system can be driven either by gravity or by pressure differential established across each component separately or across the system as a whole or across various subsets of the components. For instance, a pressure differential can be established across the cation-exchange column and the diatomaceous silica-activated charcoal column while at the same time a vacuum is drawn on the decompression chamber of the lixiviator by an appropriate arrangement of valves and pressure and vacuum lines. Such a mode of operation might be utilized, for example, when starting a second extraction in the lixiviator while the chromatography steps are being completed on a first extraction.
Lixiviator (Device IWT-113, FIG. 2)
Device IWT-113 functions to lixiviate and filter. It is made of metals, preferably a corrosion-resistant material like stainless steel. It comprises three dismountable parts, namely a xe2x80x9csealing headxe2x80x9d at the top, a xe2x80x9clixiviating barrelxe2x80x9d in the middle and a xe2x80x9cdecompressing chamberxe2x80x9d at the bottom.
The sealing head comprises a deceleration joint (1), a pressure gauge (2), a water inlet valve (3), a material filling inlet (4), a safety valve (14), a gas valve (15) and a degassing valve (16). The deceleration joint (1) transmits power to the propeller pug mill (8) by connecting a decelerating motor at a speed of 60-120 rpm so as to enable faster lixiviation and filtration by stirring. The propeller pug mill (8) can also help remove used raw material. The water inlet valve (3) is used to provide water for repeated lixiviation steps. After lixiviation, water can be supplied through the water inlet valve (3) to help pump out the used raw material from the material filling inlet while stirring.
The safety valve (14) controls the pressure inside the lixiviating barrel. The pressure is typically 0.5-1.5 kg per square centimeters while filtering. The pressure can be elevated up to 6 kg per square centimeters when necessary. The gas valve (15) maintains the pressure inside the lixiviating barrel by connecting to an air-compressing unit, whereas the degassing valve (16) functions to release the pressure inside the barrel.
The sealing head (6) is removably attached to the lixiviator. The sealing head is typically bolted on, but any removable joiner, for example a clamping device, that can seal the sealing head against the lixiviating barrel and can tolerate the pressurization can be used. A gasket for effecting a pressure seal can be inserted between the sealing head and the lixiviator barrel. The gasket material can be any typical pressure gasket material, preferably an elastomeric material that is resistant to weak acid solutions.
Filtering material (9, 10) is mounted between the decompressing chamber and the lixiviator barrel. During the lixiviation process, the filtration can be speeded by elevating the pressure in the lixiviation barrel or reducing pressure in the decompression module. The filtering material is made from filtering paper, filtering cloth and other porous filtering material that is compatible with use between stainless steel components. xe2x80x9cCompatible with use between stainless steel componentsxe2x80x9d means that the filter material will at least not promote electromotive corrosion of the stainless steel.
The filtering material can be for example, a nylon mesh having 100 to 200 meshes per square inch, a stainless steel mesh having 40 to 60 meshes per square inch, or a porous metal plate having a pore diameter of from 2 to 10 mm, preferably from 2 to 4 mm, more preferably 2.5 to 3.5 mm. These materials can be combined in a layered arrangement. Thus, the filter material can be a combination for example, of a nylon mesh having 100 to 200 meshes per square inch, middle to high speed filtering paper, a stainless steel mesh having 40 to 60 meshes per square inch, and a porous metal plate having a pore diameter of 3 mm. A preferred arrangement is to have a metal plate on the bottom, upon which is stacked a stainless steel mesh, a filtering paper and then nylon mesh on top.
The filter is preferably installed in a frame that can be removed from the lixivating/filtering apparatus for easy cleaning and maintenance. Also, the filter is preferably mounted in an elastomeric material suitable for maintaining a pressure seal between the filter and the walls of the lixiviator barrel and/or decompression chamber.
A vacuum valve (13) is installed on the side of the decompression chamber, and a drain (18) at the bottom.
The lixiviator can be mounted on a stand (19).
A heater can be fitted to the lixiviator. The heater should be capable of heating the liquid to a desired temperature and maintaining that temperature. The heater can be an oil-bath heater or steam heater of industry standards. Alternatively, the filtered solution collecting in the decompressing chamber at the bottom of the lixiviator can be passed to a separate heated vessel.
A second filtering apparatus is connected to the outlet of the lixiviator or of the separate heated vessel. The second filtering apparatus can be integral with the separate heated vessel. The filtering material of the second filtering apparatus functions to remove precipitate from the heated filtrate. The filtering material of the second filtering apparatus can be made from material similar to that of the filter between the lixiviator barrel and decompression chamber of the lixiviator. However, the filtering material of the second filtering apparatus is more preferably one having a smaller pore size, sufficient to remove small bits of precipitated protein so that such precipitate does not interfere with subsequent chromatographic steps. The filtering material for the second filtering apparatus preferably includes a middle to high speed filtering paper, in order to separate out the smaller bits of precipitated protein. The order of the filtering material components is the same as that of the first filter. Also, rather than xe2x80x9cmeshxe2x80x9d type filtering material, an membrane type filter, typically made from nylon or other polymeric material, suitable for filtration driven by a pressure differential is preferred as the filter material for the second filtration apparatus.
The second filtering apparatus is preferably operable by pressure differential and therefore the second filtering apparatus and the separate heated vessel may comprise appropriate connections to pressure or vacuum lines and appropriate relief valves as needed.
Ion-exchange Column (Device IWT-312)
An ion-exchange column (IWT-312) is used to remove proteins, peptides and their derivatives from the tetrodotoxin solution during enrichment and purification of tetrodotoxin. If not removed, these substances produce a high viscosity solution and also interfere with precipitation of the tetrodotoxin later in processing.
Diatomaceous Silica-active Charcoal Column (Device IWT-412)
A column comprising diatomaceous silica and activated charcoal is used to remove basic amino acids remaining in the eluate from the ion-exchange column.